Industrial Microbiology

INDM 4005

Lecture 10

24/02/04

4. INFLUENCE OF PROCESS VARIABLES

Overview

• Nutrient Limitation

• Cell Immobilisation

4. Influence of process variables

4.1. Kinetics and technology of nutrient limitation

4.1.1. Types of continuous culture;

4.1.2. Kinetics of continuous culture;

4.1.3. Typical pattern of biomass and substrate levels in continuous culture fermenter

4.1.4. Influence of growth constants on biomass behaviour in continuous culture

4.1.5. Application of continuous culture;

4.1.6. Advantages / disadvantages of continuous culture

4.1.7. Modifications of basic chemostat;

4.2. Nutrient limitation also applied in fed-batch

4.2.1. Fed-batch

4.2.2. Industrial application of fed-batch

4.3. Nutrient limitation and cell composition

4.4. Use of continuous culture for calculation of growth kinetics

Overview

Batch Cultures

• Closed systems microorganisms undergo a predictable

pattern of growth characterised by 4 phases

• Describe the 4 phases of growth and the factors

influencing them

• Understand the mathematics of exponential growth

• Define and apply growth parameters (td, m, mmax, k, Ys)

• Describe the Monod relationship and the meaning of ks

4.1. KINETICS AND TECHNOLOGY

OF NUTRIENT LIMITATION

Type of culture;

Batch; m varies during culture cycle

Fed-batch; m is controlled or regulated after a

certain time

Continuous; m is controlled

m reflects the physiological state or intracellular

environment

control m control intracellular environment

Growth in Continuous Culture

• Scientists are trained to conduct experiments in

which only one variable is changed at any one

time

• Continuous culture methods enable constant cell

numbers to be maintained in a constant

chemical environment at specified growth rates

for prolonged periods of time

• In this lecture we will focus on the theoretical

and practical aspects of growth in flow-through

systems

Overflow

Effluent

Fresh medium from

reservoir

Sterile air Flow-rate

regulator

Stirrer

Culture

Set up for Continuous culture

4.1.1. TYPES OF CONTINUOUS CULTURE

Method of control;

Chemostat - regulated by control of

concentration of limiting nutrient

Turbidostat - regulated by biomass using

optical density (photoelectric cell)

Biostat - regulated by systems monitoring

biomass other than optical density (e.g CO2

production)

How can the population density and

growth rate be controlled?

• To regulate the growth rate and density it is necessary to

control the influx of nutrients per unit time

• A distinctive feature of a chemostat is that one nutrient (C,

N, P, energy source, growth factor) is at a low conc

• By selecting the concentration of substrate we can

predetermine a certain microbial growth rate

• After a period of adjustment a steady-state equilibrium is

achieved

• Changing the initial substrate concentrations alters the

population density but growth rate remains unaltered at

the new steady-state

Fermenter configuration

STR

Up-flow

Plugflow

Single-stage

Multi-stage

Cell recycle

Draw diagrams and make notes on the above

CASE STUDY

Re Continuous Culture draw a diagram of a typical

pilot/ laboratory system and an industrial system

Cell

Number

Time in Hours

Steady State

The development of growth in a chemostat

Inoculation mmax

Population density increases

Nutrient limitation causes decrease in m

Growth rate equals loss of cell biomass

Mathematical relationships of

growth in chemostats

• Relationship between growth rate or specific

growth rate and medium flow can be

described mathematically

• The medium flow through the system is

represented by the term dilution rate

D =

D = dilution rate (h-1)

V = culture volume (l)

F = flow rate (l h-1)

F

V

4.1.2. KINETICS OF CONTINUOUS CULTURE

Thus:

• Mass balance or the rate of change of cells in reactor = RATE of

ACCUMULATION minus RATE of LOSS

dX /dt = m.X - D.x

Mass balance of the substrate = INPUT minus LOSS DUE TO

OUTFLOW minus SUBSTRATE USED BY BIOMASS

dS / dt = D. Sr - D. S - m. X / Y X = Total biomass

D = Dilution rate

x = Biomass concentration

m = Specific growth rate

Y = Yield

S = Substrate conc in fermenter

Sr = Substrate conc in reservoir

• The empirically derived equation for the relationship between

specific growth rate and [S] is Monod equation

D = m max . S / (Ks + S)

This is the most basic model for continuous culture

NOTE; When dX / dt = 0 (at steady state) then D = m

• This equation demonstrates how the steady state substrate

concentration in the chemostat is determined by the dilution

rate

INCORPORATE MONOD MODEL

Exponential phase Chemostat

of batch culture operating in

steady-state

Growth rate of culture

Specific growth rate of culture

Biomass

Available nutrients

Culture volume

Toxic metabolites

Constant, Variable, Increasing, Decreasing

Increasing

Constant

Increasing

Decreasing

Constant

Increasing

Constant

Constant

Constant

Constant

Constant

Constant

Batch versus Chemostat

CASE STUDY

A chemostat operating in steady-state at a

dilution rate of 0.25 h-1 sets a limiting nutrient

concentration of 0.6 micromoles l-1. Determine

the Monod constant in suitable units if mmax for

the organism is 0.25 h-1

D = m max . S / (Ks + S)

Rearrange the equation

m max - D

Ks = s

D

(0.6 - 0.25)

Ks = 0.6

0.25

Ks = 0.6 x 1.4

Ks = 0.84 micromoles l-1

THE PERFECT MODEL WOULD REQUIRE AN UNREALISTIC

AMOUNT OF INFORMATION

Simplifying assumptions are made, for example,

Assume that population density has no effect

If D = 0 then batch culture - but no lag period predicted

Transient conditions predicts either stable condition or

wash-out

Assumes all substrate goes to biomass (maintenance!)

No allowance for substrate or product inhibition

In more advanced models these areas must be considered

4.1.3. TYPICAL PATTERN OF BIOMASS AND

SUBSTRATE LEVELS IN CONTINUOUS

CULTURE FERMENTER

CASE STUDY

Plot; steady state substrate concentration

steady state biomass concentration

steady state product concentration

against dilution rate (m) Page 15 Stanbury & Whitaker

4.1.4. INFLUENCE OF GROWTH CONSTANTS

ON BIOMASS

BEHAVIOUR IN CONTINUOUS CULTURE

Influence of low vs high Ks or mmax on biomass or substrate level

Influence of low vs high Ks or mmax of different populations on

competition

DEVIATIONS FROM IDEAL BEHAVIOUR may be due to

Maintenance energy

Synthesis of reserve polymers

Switch to less efficient pathways

Imperfect mixing

Substrate toxicity

Second substrate becomes limiting

4.1.5. APPLICATION OF

CONTINUOUS CULTURE

INDUSTRY;

• Waste-treatment

• Single-cell protein

• Continuous beer production

• Continuous amino acids, organic acids production

• Continuous ethanol

• Continuous bakers yeast

RESEARCH - more important

• Physiology and biochemistry growth rate control

Influence of environmental / process factors on growth and product

formation.

Induction, repression, growth rate, influence of temperature, pH etc.

• Microbial ecology

Selection of slow growing populations

Prey-predator interactions

Competition (e.g plasmid +/-)

• Kinetics

Calculation of growth constants, fermentation data

CASE STUDY

From the literature record some applications of

continuous culture to studies in microbial physiology

and ecology

4.1.6 ADVANTAGES / DISADVANTAGES OF CC

Advantages

• Uniformity of operation

• Process demands are constant

i.e. continuous cycle of sterilisation, fermentation, harvesting, extraction

• Once in steady-state demands re process control are constant

i.e. oxygen demand

Disadvantages

• Susceptibility to contamination

• Duration of run is longer increased chance of contamination

• Strain degeneration arising from large number of generations

• Contamination with "fitter" competitor e.g. lower Ks

OBJECTIVES IN INDUSTRIAL

APPLICATION?

CONTINUOUS PROCESSING

Advantage ?

example beer Residence time of "pint" in brewery

same.

example waste-treatment definite advantage.

EXERT PHYSIOLOGICAL CONTROL

Can use fed-batch which is less demanding

4.1.7. MODIFICATIONS OF BASIC

CHEMOSTAT

• MULTI-STAGE

Different environments or growth rates in the various reactors (e.g.

1st biomass, 2nd product)

• SINGLE STAGE WITH CELL RECYCLE

Application in activated sludge waste-treatment

Relationship between D and m different when recycle used.

EFFECT OF FEEDBACK;

1. Increase biomass conc. in fermenter - lower in effluent

2. Decrease residual substrate

3. Maximise rate of product formation

4. Dcrit is increased - useful when substrate is dilute

F1

SR

X1

S1

V1

FO2

SR2

X2

S2

V2

F2

Chemostats in series

CONTINUOUS CULTURE PRINCIPLES

Also applied in;

• UP-FLOW REACTORS (often with immobilised cells)

• PLUG-FLOW SYSTEMS

4.2. NUTRIENT LIMITATION ALSO

APPLIED IN FED-BATCH

4.2.1 Fed-Batch

Takes advantage of fact that residual substrate

concentration may be maintained at very low levels

Type of continuous culture but volume is not constant.

Quasi-steady state achieved.

CLASSIFICATION OF FED-BATCH

OPERATION

• Without feed-back control - programmed feed-rate

1. Intermittent addition

2. Constant rate

3. Exponentially increased rate

• With feed-back control

1. Indirect feed-back

e.g. respiration rate, dissolved oxygen, pH

2. Direct feed-back

concentration of substrate in culture exerts control

4.2.2 INDUSTRIAL APPLICATION OF FED-BATCH

• Penicillin

Glucose, phenyl acetic acid, ammonia source

• Cephalosporin

Glucose, methionine

• Streptomycin

Glucose, ammonia source

Glutamic acid

Urea, ethanol, (acetic acid)

• Amylase

Carbon source

• Bakers Yeast

Glucose

• Citric acid

Glucose, ammonia

4.3 NUTRIENT LIMITATION and

CELL COMPOSITION

Media can be designed to allow limitation on any essential

nutrient

NUTRIENT LIMITATION EFFECT

CARBON energy supply

NITROGEN or SULPHUR protein synthesis

PHOSPHORUS Nucleic acid synthesis

MAGNESIUM or POTASSIUM Nucleic acid and or

membrane synthesis

4.3 NUTRIENT LIMITATION and

CELL COMPOSITION

THE DEGREE OF LIMITATION INFLUENCES THE

CELL COMPOSITION, for example

CELL SIZE

NUCLEIC ACIDS

CONSEQUENTLY CELLS BEHAVE DIFFERENTLY UNDER

DIFFERENT LIMITATION CONDITIONS;

Repression mechanisms may be removed, for example, antibiotic

production or pigment production under phosphate limitation

4.4. USE OF CONTINUOUS CULTURE FOR

CALCULATION OF GROWTH KINETICS

(1) Calculation of Ks and mmax

(2) Determine variation in yield with growth rate

(3) Calculation of Yg and m, endogenous respiration

(4) m /mmax to compare growth under different conditions

NOTE; growth rate becomes an independent variable in

continuous culture

4.4. USE OF CONTINUOUS CULTURE FOR

CALCULATION OF GROWTH KINETICS

• Use of higher dilution rates can lead to higher

biomass productivity

But result in

• higher substrate concentrations in the effluent and

lower biomass concentrations in the reactor due to

wash-out

• when the dilution rate exceeds the critical dilution rate

then washout occurs

4.4. USE OF CONTINUOUS CULTURE FOR

CALCULATION OF GROWTH KINETICS

• These factors have a number of consequences e.g in

continuous wastewater treatment processes

• The minimum reactor volume is set by the critical dilution rate

• High dilution rates will lead to an effluent containing high

concentration of substrate and the effluent will therefore

contain substrates/wastes and not have been treated

properly

• Low cell concentrations at high dilution rates will also make

the reactor sensitive to inhibitors in the feed. Inhibitors would

cause the specific growth rate of the cells to drop and cause

the cells to washout. The lower the conc of cells, then the

faster the cells will washout

Conclusions

• In this lecture we have seen that a chemostat is a means of providing

nutrient limitation an important process variable

• Mathematical relationships can be used to predict growth and determine

growth parameters such as mmax, Ks, Y

• List the differences between growth in batch and in continuous culture

• Understand the terms steady-state, dilution rate, growth limiting substrate,

Monod constant,

• Describe the principles of fed-batch, biomass feedback, and multi-stage

cultivation

• Give applications for continuous cultivation techniques

• Describe the main practical problems encountered in chemostat operation